Project Summary/Abstract Despite marked progress in the rate of cure among patients with acute leukemia, chromosomal translocations involving the mixed lineage leukemia gene (MLL1) give rise to highly aggressive acute leukemias associated with poor clinical outcomes for pediatric and adult patients. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged (MLL-r) leukemia in model systems. Likewise, work from the previous funding cycle clearly defined a role for DOT1L inhibition in MLL-r leukemia through characterization of a potent, specific DOT1L inhibitor, EPZ-5676 (pinometostat), that has remarkably selective anti-proliferative and pro-apoptotic effects on MLL-rearranged cells. As pinometostat has progressed through Phase I clinical trials, responses and subsequent relapses imply the development of acquired resistance to the inhibitor. In this proposal, we seek to understand the biological underpinnings of DOT1L inhibitor resistance and develop a novel class of small molecule DOT1L degraders to overcome resistance. In Specific Aim 1, we seek to understand the mechanisms of resistance through detailed studies of chromatin structure and gene expression following the development of acquired resistance to DOT1L inhibitor therapy. We will also investigate the non-enzymatic role of DOT1L in MLL-r leukemia and define dependencies acquired in the resistant state. In Specific Aim 2, we will lead a chemistry and chemical biology campaign and develop an assay platform for the identification and optimization of the first small molecule degraders of endogenous DOT1L protein. In Specific Aim 3, we will utilize our cell culture and patient-derived xenograft models of MLL-r leukemia to explore the impact of DOT1L degradation on gene expression and tumor progression while driving hit-to-lead optimization of DOT1L degraders that can prompt clinical investigation. We will also test pharmacological hypotheses regarding combinatorial drug action with DOT1L degraders in predictive human models of MLL-r leukemia. We expect these aims to bring new more efficacious, less toxic therapies to children and adults diagnosed with this devastating disease.